title: Detection and quantification of flavivirus NS5 methyl-transferase activities.
abstract: Flavivirus NS5 is the most conserved protein amongst the flavivirus proteins and is an essential enzyme for viral mRNA capping and replication. It encodes a methyl-transferase (MTase) domain at its N-terminal region which carries out sequential N7 and 2'-O methylation, resulting in the formation of the cap1 structure on its viral RNA genome. Two key methods have been established to measure these activities in vitro: thin-layer chromatography (TLC) and scintillation proximity assays (SPA). TLC offers the advantage of direct visualization of the amounts and types of cap structures formed whilst the SPA assay is more sensitive and quantitative. It is also amenable to high-throughput compound screening. The drawback of both assays is the need for radioisotope usage. We further describe the adaptation of a nonradioactive immune-competitive fluorescence polarization assay for detection of dengue virus MTase activity.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore, Singapore.
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.forename: Christophe
authors.initials: C
authors.lastname: Bodenreider
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D000818:Animals; D002855:Chromatography, Thin Layer / Q000379:methods; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D005416:Flavivirus / Q000201:enzymology*; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D012588:Scintillation Counting / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins